Asymmetric cell divisions, which produce daughter cells with different fates, are important for generating cell diversity during embryonic development, and may regulate stem cell function in many tissues (e.g. epidermis, intestine, blood, liver, germ cells, and the nervous system). Despite the clinical importance of understanding the regulation of asymmetric cell divisions, remarkably little is known about how and where asymmetric divisions occur in mammals. Our long-term goal is to identify genes controlling asymmetric cell divisions in Drosophila, and to determine if homologous murine genes control asymmetric cell divisions during embryogenesis or in adult stem cell populations. The proposed research may have clinical applications in mammalian stem cell immortalization, gene therapy, and treatment of stem cell neoplasms. We will focus on Drosophila CNS stem cells (neuroblasts), which divide asymmetrically to produce a new neuroblast and a more differentiated daughter cell (GMC). At least 4 proteins are partitioned into the daughter GMC, including the Prospero transcription factor, which is necessary for the transition from neuroblast-specific to GMC-specific gene expression. The specific aims of this proposal are: (1) To identify new genes regulating neuroblast asymmetric cell divisions. We will screen for mis-localization of Prospero protein, and characterize mutants by standard molecular genetic methods. (2) To characterize miranda, a gene encoding a novel coiled-coil protein required for the asymmetric localization of Prospero in neuroblasts. We will sequence protein-positive miranda EMS alleles that affect Miranda localization (3 alleles) or Prospero binding (2 alleles) to map each functional domain; use a yeast two hybrid screen to identify proteins specifically binding the Miranda localization domain; screen for novel Miranda- binding "cargo" proteins; and determine the structure of Miranda functional domains (with Dr. M. Churchill). (3) To further characterize prospero function. We will determine if prospero RNA translational repression in neuroblasts (but not GMCs) is necessary to establish distinct sibling fates; assay the role of phosphorylation in Prospero localization; and assay prospero expression and function in the adult sensory nervous system. (4) To isolate murine homologues of miranda (and genes identified in Specific Aim 1), and to characterize their role in regulating asymmetric divisions during embryogenesis and in adult stem cells. We will do RNA and protein localization studies; our collaborator, Dr. G. Oliver will generate and assay the gene knock-out mice.